Role of Calcium Signals on Hydrogen Peroxide-Induced Apoptosis in Human Myeloid HL-60 Cells.

The present study is aimed to determine the role of calcium signaling evoked by the oxygen radical, hydrogen peroxide (H2O2) and the specific inhibitor of calcium reuptake thapsigargin on apoptosis in the human leukemia cell line HL-60. Our results show that treatment of HL-60 cells with 100 µM H2O2 and 1 µM thapsigargin induced a transient increase in cytosolic free calcium concentration ([Ca(2+)]c) due to calcium release from internal stores. These stimulatory effects on calcium signals were followed by activation of the mitochondrial permeability transition pore (mPTP), as well as a time-dependent increase in caspase-9 and -3 activities. Our results also show that H2O2 and thapsigargin were able to increase the relative content of fragmented DNA and phosphatidylserine externalization, as detected by double-staining with propidium iodide (PI) and annexin-V-FITC, respectively. Treatment of cells with H2O2 or thapsigargin resulted in activation of the proapoptotic protein Bid. Furthermore, coimmunoprecipitation experiments showed active Bax was bound to Bid, which regulates Bid activity and promotes apoptosis. Our findings suggest that H2O2(-) and thapsigargin-induced apoptosis is dependent on rises in [Ca(2+)]c in human myeloid HL-60 cells.


INtrODUctION
Apoptosis is a gene-regulated form of cell death that is critical for normal development and tissue homeostasis. A major component of the apoptotic machinery involves a family of aspartic acid-directed cysteine proteases, called caspases (cysteinyl aspartate-specific proteinases), which cleave multiple protein substrates en masse, leading to the loss of cellular structure and function and ultimately re-sulting in cell death (1).
From a functional point of view, caspases are involved in apoptosis acting both initiators (caspase 8, 9, and 10) and effectors (caspase 3, 6, and 7) (2). Caspase 8 was identified as the most important initiator enzyme of the Fas/ CD95 pathway (3). Caspase 9 interacts with many other regulators and transducers, such as cytochrome c, in intrinsic pathway (4). Both initiator caspases are activators of downstream caspases. Caspase 3, the most important among them, executes the final disassembly of the cell by cleaving a variety of cell structure proteins and generating DNA strand breaks (5).
Traditionally, two apoptotic pathways have been described. One pathway is the so-called extrinsic pathway initiated by the binding of an extracellular death ligand, such as FasL, to its cell-surface death receptor, such as Fas (6). The second pathway is the intrinsic pathway, which is mediated by mitochondrial alterations. In response to ORIGINAL ARTICLE apoptotic stimuli, several proteins are released from the intermembrane space of mitochondria into the cytoplasm (7). Some of these well-characterized proteins include cytochrome c, which mediates the activation of caspase-9 (8), triggering in turn a cascade of caspase activation, including caspase-3, which promotes cellular self-destruction.
On the other hand, whereas ion calcium is a key regulator of cell survival, the sustained elevation of cytosolic free calcium concentration ([Ca 2+ ] c ) plays a role in cell death (9). The proapoptotic effects of calcium are mediated by a diverse range of calcium-sensitive factors that are compartmentalized in various intracellular organelles including endoplasmic reticulum and mitochondria (10). Excessive calcium load to the mitochondria may induce apoptosis by stimulating the release of apoptosis promoting factors from the mitochondrial intermembrane space to the cytoplasm and by impairing mitochondrial function (11).
Calcium-dependent increase in mitochondrial permeability to ions and solutes with molecular masses up to 1500 Da, matrix swelling, and uncoupling of oxidative phosphorylation, which has been defined as permeability transition (12), is currently ascribed to the opening of the mitochondrial permeability transition pore (mPTP) (13). The mPTP is a megachannel formed in the mitochondrial membranes which is found at the contact sites between inner and outer mitochondrial membranes (14,15). Under conditions like oxidative stress, calcium overload, and low ATP levels, a number of members of Bcl-2 family are recruited to enable the pore formation at its high conductance state (14,15).
Furthermore, mitochondria are the major source of reactive oxygen species (ROS) in the cell.
is generated by the operation of complexes I and III in the matrix and is converted to hydrogen peroxide (H 2 O 2 ) by Mn-superoxide dismutase. Apoptosis can also be stimulated by ROS in several cell types (16)(17)(18). Recently, it has been reported that ROS can mediate the apoptosis induced by either growth factors, such as transforming growth factor-β (TGF-β) in human lens epithelial cells (19), or classical agonists, such as thrombin in human platelets (20) or progesterone in human spermatozoa (21).
Although ROS and calcium have been separately reported to be important mediators of apoptosis, little correlation between these two mediators has been reported. Here, we focused on the role of intracellular calcium in H 2 O 2 -induced apoptosis in human leukemia cell line HL-60 in culture, analyzing caspase-3 and -9 activation, mitochondrial depolarization, induction of the mitochondrial permeability transition pore and activation of the proapoptotic proteins Bax and Bid.

cellular culture
Human promyelocytic leukemia HL-60 cells were grown in RPMI 1640 medium supplemented with 2 mM L-glutamine, 10% heat-inactivated fetal bovine serum, 100 U/mL penicillin, 100 µg/mL streptomycin and 1.25% DMSO at 37ºC under a humidified condition of 95% air and 5% CO 2 . Cells were routinely plated at a density of 3 × 10 5 cells/ml into fresh flasks and the viability was >95% in all experiments as assayed by the trypan-blue exclusion method.

Assay for caspase activity
To determine caspase-3 and -9 activity, stimulated or resting cells were sonicated and cell lysates were incubated with 2 mL of substrate solution (20 mM HEPES, pH 7.4, 2 mM EDTA, 0.1% CHAPS, 5 mM DTT and 8.25 µM of caspase substrate) for 1 h at 37 ºC as previously described (24). The activities of caspase-3 and -9 were calculated from the cleavage of the respective specific fluorogenic substrate (AC-DEVD-AMC for caspase-3 and AC-LEHD-AMC for caspase-9). Substrate cleavage was measured with a fluorescence spectrophotometer with excitation wavelength of 360 nm and emission at 460 nm. Preliminary experiments reported that caspase-3 or -9 substrate cleaving was not detected in the presence of the inhibitors of caspase-3 or -9, DEVD-CMK or z-LEHD-FMK, respectively. The data were calculated as fluorescence units/mg protein and presented as fold increase over the pretreatment level (experimental/control).

calcein and cl 2 co co-loading
Cells were loaded with 1 µM calcein-AM in the presence of 1 mM Cl 2 Co during 15 minutes at 37ºC and later were washed using always the same RPMI medium where the cells had been grown. Mitochondrial calcein fluorescence was measured with a fluorescence spectrophotometer with excitation wavelength of 488 nm and emission at 515 nm (25). Data are expressed as fractional changes of emitted fluorescence (F/F 0 ).

Annexin-V staining
Cells were harvested through and washed twice with PBS and centrifuged at 500 g for 5 min, then the supernatant was discarded and the pellet was resuspended in 95 μL annexin-V-binding buffer at a density of 10 5 -10 6 cells/ mL with 2.5 µg/mL annexin-V-FITC at 4ºC for 30 min. Cells were analyzed by flow cytometry on a FACScan cytometer (BD Biosciences) after addition of 2.5 µg/ mL propidium iodide (PI). 10,000 events were analyzed using the FL-1 and FL-3 detector filters. Each sample was tested 3 to 5 times in independent experiments. Annexin-V binds to those cells that express phosphatidylserine on the outer layer of the cell membrane, and PI stains the cellular DNA of those cells with a compromised cell membrane. This allows for live cells (unstained with either fluorochrome) to be discriminated from apoptotic cells (stained only with annexin-V) and necrotic cells (stained with both annexin-V and PI).

cell cycle analysis
After treatment, cells (approximately 1 × 10 6 cells/mL) were washed with PBS and fixed in 70% ethanol for 30 min at 4ºC. The cells were again rinsed with PBS and resuspended in 500 µL PBS containing 2.5 µg/mL PI and 50 µg/mL RNase. The samples were kept in the dark at 4ºC for 30 min and analyzed by flow cytometry on a FACScan cytometer (BD Biosciences) using the FL-2 detector filter. Cells undergoing apoptosis stain with PI and exhibit a reduced DNA content with a peak in the hypodiploid region (26) The percentages of every phase (G2/M, S, and G0/G1) were analyzed and the percentage of apoptosis was taken as the fraction with hypodiploid DNA content.

Immunoprecipitation
Bax activation and association with Bid were determined by immunoprecipitation as previously described (27). Briefly, HL-60 cell suspensions were stimulated, as indicated, and lyzed by mixing equal volume of cell sample with a radio immunoprecipitation assay buffer (RIPA 2x). Following preparation of cell lysates, protein concentration was determined using the Bradford assay and adjusted to a protein concentration of 50 µg/mL with lysis buffer. Bax was immunoprecipitated from cell lysates by incubation with 2 μg of anti-Bax antibody (clone 6A7) and protein A-agarose overnight at 4°C on a rocking platform. Immunoprecipitates were resolved by 15% SDS-PAGE and Western blotting was performed as described in the following section.

Western blotting
Bax and Bid expression in HL-60 cells was analyzed by Western Blotting. Briefly, HL-60 cell suspensions were stimulated, as indicated, and lyzed by mixing with the appropriate amount of Laemmli buffer. Following preparation of cell lysates, protein concentration was determined using the Bradford assay and adjusted to a protein concentration of 50 µg/mL with lysis buffer. Proteins were separated by 15% SDS-PAGE and electrophoretically transferred, for 2 h at 0.8 mA/cm 2 , in a semi-dry blotter (Hoefer Scientific, Newcastle, Staffs., U.K.) onto nitrocellulose for subsequent Western blotting. Non-specific protein binding sites of the nitrocellulose membranes were blocked by incubating overnight with 10% (w/v) BSA in Tris-buffered saline with 0.1% Tween 20 (TBST). Membranes were incubated with the anti-Bax antibody diluted 1:200 in TBST for 2 h or with the anti-Bid antibody diluted 1:500 overnight. To detect the primary antibodies, blots were incubated with horseradish peroxidase-conjugated sheep anti-mouse IgG antibody diluted 1:5000 or horseradish peroxidase-conjugated donkey anti-rabbit IgG antibody diluted 1:10000 in TBST, respectively, and exposed to enhanced chemiluminescence reagents for 5 min. Blots were then exposed to photographic films and the optical density was estimated using scanning densitometry.

statistical Analysis
Data are presented as mean ± standard error of mean (S.E.M.) and analysis of statistical significance was performed using Student's t-test. p<0.05 was considered to indicate a statistically significant difference.

Effects of H 2 O 2 on calcium mobilization
In the presence of normal extracellular calcium concentration, fura-2-loaded HL-60 cells were treated with the oxygen radical H 2 O 2 . As shown in Figure 1A, stimulation with 100 µM H 2 O 2 caused a slow and sustained [Ca 2+ ] c increase, which reached a stable [Ca 2+ ] c plateau after 5-7 minutes of administration. Figure 1 also demonstrates that the increase of [Ca 2+ ] c induced by 100 µM H 2 O 2 was also observed in calcium-free medium ( Figure 1B), reflecting the release of calcium from intracellular store(s), though the calcium signal was smaller compared to that obtained in the presence of extracellular calcium. As expected,  Figure  1C). This increase induced by thapsigargin was also observed in a calcium-free medium ( Figure 1D), also reflecting the release of calcium from internal pools.

H 2 O 2 induces activation of caspase-3 and -9
To examine the effect of H 2 O 2 on caspase-3 activation, HL-60 cells were treated with 100 µM H 2 O 2 for 10-120 min. As shown in Figure 2A, treatment of cells with 100 µM H 2 O 2 induced a time-dependent activation of caspase-3. Cell stimulation with 100 µM H 2 O 2 caused a de-tectable activation of caspase-3 after 20 min of treatment with a 134 ± 12 % above control, and the maximum effect was obtained after 120 min of stimulation (215 ± 31 % above control, Figure 2A, p<0.05). Caspase-9 is an initiator caspase that is involved in the initial steps of mitochondrial apoptosis (9). To investigate whether the activation of caspase-3 induced by H 2 O 2 is a mitochondrial apoptosis, we checked the caspase-9 activity in the presence of 100 µM H 2 O 2 . Consistent with the results presented above, activation of caspase-9 by 100 µM H 2 O 2 showed a similar activation pattern to that of caspase-3, reaching a maximum peak of caspase-9 activity after 120 min of stimulation, with a 189 ± 40 % above control ( Figure 3A).
However, the effect of H 2 O 2 on caspase-3 and -9 activation was not clearly concentration-dependent, as shown  in Figures 2B and 3B. In fact, treatment for 120 min at 1 µM or 10 µM was unable to induce activation of caspase-3 and -9. The maximun effect was obtained with 100 µM of H 2 O 2 , while 1 mM H 2 O 2 induced a lower activation of the caspase-3 and -9 than 100 µM H 2 O 2 ( Figure 2B and 3B). In addition, the treatment of HL-60 cells with 1 µM thapsigargin, a sarco-endoplasmic reticulum calcium ATPase (SERCA) inhibitor, which depletes the intracellular calcium stores, was also able to induce a time-dependent activation of caspase-3 and -9, reaching a maximal caspases activities after 60 minutes of stimulation ( Figure 4).

Activation of mPtP by H 2 O 2
To further investigate the role of H 2 O 2 in mitochondrial apoptosis, we probed mPTP opening in HL-60 cells loaded with calcein-AM in the presence of cobalt chloride to quench fluorescence from all cellular domains except from within mitochondria (25,30). Using this protocol, 100 µM H 2 O 2 caused a loss of mitochondrial calcein fluorescence ( Figure 5). Similar results, although to a larger extent, were obtained when the cells were treated with 1 µM thapsigargin ( Figure 5), suggesting that H 2 O 2 and thapsigargin cause permeability transition pore induction in mitochondria. Although this protocol does not distinguish between calcein efflux and cobalt influx, it is consistent with induction of the permeability transition pore.

Effects of H 2 O 2 on cell cycle
To evaluate the effect of H 2 O 2 on cell cycle, cells were stained with PI which has affinity for nucleic acids, and incubated with RNase in presence of triton X-100, 0.01% as nonionic detergent which supplies the PI staining. An increase in hypodiploid DNA content was found in H 2 O 2treated cells. Treatment with 100 µM H 2 O 2 for 120 minutes showed an important Sub-G1 arrest at expense of the rest of the phases, mainly G0/G1 was decreased after the H 2 O 2 treatment compared with the control group ( Figure 6A). On the other hand, treatment of cells with 1 µM thapsigargin for 60 minutes shows similar results, which consisted in an increase of sub-G1 and a decrease in other phases compared with the control group ( Figure 6A).
Annexin-V-FITC and PI stainings were used to detect apoptotic and necrotic changes in H 2 O 2 -treated HL-60  cells. Double-stained cells were analyzed by flow cytometry and the results revealed that a 9 ± 1.5 % of control cells had only annexin-V positivity (early apoptotic cells) and 4 ± 1 % annexin-V/PI positivity (necrotic cells) ( Figure 6B). As also shown in Figure 6B, 100 µM H 2 O 2 induced an increase in the percentage of apoptosis. The population of early apoptotic cells increased after 120 minutes treatment with 100 µM H 2 O 2 (28 ± 0.5%, Figure 6B). In addition, the treatment of cells with 100 µM H 2 O 2 for 120 minutes induced a slight increase in the population of necrotic cells as compared with the control group (7 ± 0.6 % versus 4 ± 1%). For comparison, the effect of administration of 1 μM thapsigargin for 60 minutes is included in Figure 6. Thapsigargin treatment increased in the same way the number of apoptotic cells in a 29 ± 1%, as revealed by annexin-V assay, and the percentage of necrotic cells was 7 ± 0.7% ( Figure 6B). These results indicate that the treatments with H 2 O 2 and thapsigargin trigger cells to apoptosis.

Effects of H 2 O 2 on activation of bax and bid
We have further investigated whether the expression of the proapototic proteins, Bax and Bid may be altered by treatment of HL-60 cells with H 2 O 2 . Bid activation was analyzed by Western blotting using a rabbit anti-Bid antibody that detects both the cleaved (active) and native forms of Bid (31). Active Bax was detected by immunoprecipitation with the anti-Bax antibody (clone 6A7), which reacts only with Bax in its conformationally active state, followed by Western blotting with the same antibody as described previously (27,32). Other forms of Bax were detected by Western blotting of the samples with the anti-Bax antibody (clone 6A7). As shown in Figure 7A, 100 µM H 2 O 2 and 1 µM thapsigargin did not significantly modify the expression of Bax in these cells, detected as the sum of the amount of the forms of Bax detected at 42 and 21 kDa (after treatment with H 2 O 2 or thapsigargin Bax expression increased by 106.3 ± 9.8% of control and 109.2 ± 10.6% of control, respectively). In contrast, Bid expression, estimated as described for Bax, was significant increased after treatment with both agents, with an increase of 117.0 ± 6.3% of control after treatment with 100 µM H 2 O 2 and 111.1 ± 1.2% of control in cells treated with thapsigargin (p<0.05, Figure 7C). As shown in Figure 7B, incubation of HL-60 cells with either 100 µM H 2 O 2 or 1 µM thapsigargin slightly enhanced Bax activity by 102.1 ± 2.1% of control and 103.6 ± 8.2% of control, respectively. On the other hand, we also analyzed Bax activation and Bax/Bid association by co-immunoprecipitation using a mouse monoclonal anti-Bax antibody (clone 6A7) that recognises a specific region that is very close to the Bax dimerization domain that is only exposed once Bax is activated in its monomeric conformation (33). We found that HL-60 treatment with either 100 µM H 2 O 2 for 120 minutes or 1 µM thapsigargin for 60 minutes, significantly increased Bid and Bax association in a 149.7 ± 28.6% of control and in a 147.1 ± 35.0% of control, respectively ( Figure 7D, p<0.05).

DIscUssION
The proapoptotic effects of calcium are mediated by a diverse range of calcium-sensitive factors that are compartmentalized in various intracellular organelles including endoplasmic reticulum and mitochondria (28). It has been reported that a prolonged elevation in [Ca 2+ ] c and alterations in calcium homeostasis initiate the mitochondrial apoptotic pathway (9, 28) and induce endoplasmic reticulum stress that, in turn, leads to apoptosis (29). Excessive calcium load to the mitochondria may induce apoptosis by stimulating the release of apoptosis promoting factors from the mitochondrial intermembrane space to the cytoplasm and by impairing mitochondrial function (11,34). Apoptosis can also be stimulated by ROS in several cell types (24)(25)(26); even it has been shown that H 2 O 2 evokes increased [Ca 2+ ] c in the absence of extracellular calcium, indicating that H 2 O 2 mobilizes calcium from intracellular stores (7,21) leading cells into an apototic state.
This study was designed to determine the effects of the reactive oxygen species H 2 O 2 and the specific inhibitor of calcium reuptake thapsigargin on mitochondrial apoptosis in the HL-60 tumor cell line, which is a useful cellular model to study cell growth of leukemia cells. Our results demonstrate that H 2 O 2 and thapsigargin evoked apoptotic events through the increase of [Ca 2+ ] c in HL-60 cells, inducing caspase -9 and -3 activation, induction of the mPTP and activation of proapoptotic proteins.
Caspase-9 and -3 are major mediators of apoptotic cell death and their activation is widely considered an apoptotic marker. Here, we show that stimulation of HL-60 cells with H 2 O 2 or thapsigargin increased caspase-3 activity in a time-dependent manner. These results are in agreement with previous studies, where the activation of executioner caspase-3 is reported by H 2 O 2 and thapsigargin (35)(36)(37). According to Shi et al. (4) caspase-3 can be also activated by either extrinsic and by mitochondrial-mediated (intrinsic) pathway. Additionally, our results also demonstrate a time dependent activation of the initiator caspase-9 induced by administration of H 2 O 2 or thapsigargin, which is indicating the mitochondrial involvement in the activation of caspase-3. In addition, our findings demonstrate that caspase activation evoked by H 2 O 2 or thapsigargin is accompanied by induction of mPTP, which strongly suggests the mitochondrial participation of the intrinsic pathway of apoptosis. Our results are in agreement with studies which support that endogenous reactive oxygen species, such as H 2 O 2 , cause apoptosis through mPTP (38,39), and an excessive intracellular [Ca 2+ ] has been linked to activation of the mPTP and induction of apoptosis (40).
A number of studies showed that high concentration of H 2 O 2 induces necrosis and low concentration induces apoptosis (41)(42)(43). Although apoptosis and necrosis have different effects on cellular physiology, the cellular response to H 2 O 2 is continuum from apoptosis to necrosis (41). Our findings by flow cytometry indicate that the treatment of H 2 O 2 for 120 minutes induces a significant increase in both the population of apoptotic cells and the population of necrotic cells, but the increase in the apoptotic population was notably higher than the necrotic one. Similar results were found when the intracellular calcium stores were depleted by thapsigargin, which showed a higher increase in the annexin-V stained population than double stained one (annexin-V and PI). This suggests that depletion of intracellular calcium stores by thapsigargin or oxidative stress resulting from H 2 O 2 treatment induce early apoptotic state mainly in HL-60 cells. Alternatively, in this study we showed that the effects of H 2 O 2 and thapsigargin on caspase activation are related with alteration in the cell cycle progression. In fact, depletion of intracellular calcium stores by H 2 O 2 and thapsigargin reveals variations on cell cycle since the G2/M and S phases are arrested by the Sub-G1 one. This result indicates that H 2 O 2 and thapsigargin have proapoptotic effects.
During apoptosis several members of the Bcl-2 family undergo activation. For instance, caspase 8 activation, via stimulation of Fas in the plasma membrane, induces rapid activation of Bid and evokes its translocation to mitochondria as tBid. tBid, with 15 kDa, is able to bind Bax favoring their anchoring to the mitochondrial membrane, a process that is required for the activation of MAP-1 and the release of cytochrome c (44)(45)(46). Bid activation was analyzed by Western blotting using a rabbit anti-Bid antibody that detects both the cleaved (active) and native forms of Bid (31). Active Bax was detected by immunoprecipitation with the anti-Bax antibody (clone 6A7), which reacts only with Bax in its conformationally active state, followed by Western blotting with the same antibody as described previously (27,32). Other forms of Bax were detected by Western blotting of the samples with the anti-Bax antibody (clone 6A7). Our results indicate that neither H 2 O 2 nor thapsigargin induce Bax activation; however, association of Bax and Bid is induced by both H 2 O 2 and thapsigargin, as well as Bid activation under the same treatments in HL-60 cells. The effects of the treatments in HL-60 cells are in agreement with Lee (47) who probed that H 2 O 2 is not able to alter Bax activity, or even, other anticancer drugs that have been evaluated in this cell model, such as furanodiene, which is only able to alter Bid activity, meanwhile other Bcl-2 family members like Bax or Bcl-2 remain unaltered (48). However, other results suggest that Bax is activated and translocated to the mitochondria by H 2 O 2 in SW480 human colon adenocarcinoma cell line (49). This apparent discrepancy could be attributed to the different methodological procedures to determine Bax activation. In the present study we stimulated the cells with 100 µM for 120 minutes, while results shows by Nie et al. (49) were obtained after 6 hours of incubation, whereas under short time (6 hours) the endogenous Bax proapoptotic activity was not detected (49). All together, our results suggest that not only proapoptotic proteins Bax and Bid are less active in resting HL-60 cells than in cells where calcium reuptake has been inhibited, or in cells treated with ROS, but also that cell treatment with both agents induced activation of the transduction and expression mechanism of both proapoptotic proteins, which eventually resulted in a cellular death by apoptosis.

cONcLUsIONs
The data presented here show the oxygen radical H 2 O 2 and the inhibitor of calcium reuptake thapsigargin cause activation of caspase-3 and -9. Our studies also demonstrate that apoptotic events induced by both treatments are mainly of mitochondrial origin and are able to increase cell death by apoptosis and necrosis. We conclude that intracellular calcium depletion by treatment with H 2 O 2 or thapsigargin, may induce the mitochondrial calciumoverload which in turn would generate more ROS which activate apoptotic phenomenon in the human myeloid cell line HL-60.